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Apoptotic Genes As Potential Markers of Metastatic Phenotype in Human Osteosarcoma Cell Lines

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Apoptotic Genes As Potential Markers of Metastatic Phenotype in Human Osteosarcoma Cell Lines 17-31 10/12/07 14:53 Page 17 INTERNATIONAL JOURNAL OF ONCOLOGY 32: 17-31, 2008 17 Apoptotic genes as potential markers of metastatic phenotype in human osteosarcoma cell lines CINZIA ZUCCHINI1, ANNA ROCCHI2, MARIA CRISTINA MANARA2, PAOLA DE SANCTIS1, CRISTINA CAPANNI3, MICHELE BIANCHINI1, PAOLO CARINCI1, KATIA SCOTLANDI2 and LUISA VALVASSORI1 1Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna; 2Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli; 3IGM-CNR, Unit of Bologna, c/o Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy Received May 29, 2007; Accepted July 19, 2007 Abstract. Metastasis is the most frequent cause of death among malignant primitive bone tumor, usually developing in children patients with osteosarcoma. We have previously demonstrated and adolescents, with a high tendency to metastasize (2). in independent experiments that the forced expression of Metastases in osteosarcoma patients spread through peripheral L/B/K ALP and CD99 in U-2 OS osteosarcoma cell lines blood very early and colonize primarily the lung, and later markedly reduces the metastatic ability of these cancer cells. other skeleton districts (3). Since disseminated hidden micro- This behavior makes these cell lines a useful model to assess metastases are present in 80-90% of OS patients at the time the intersection of multiple and independent gene expression of diagnosis, the identification of markers of invasiveness signatures concerning the biological problem of dissemination. and metastasis forms a target of paramount importance in With the aim to characterize a common transcriptional profile planning the treatment of osteosarcoma lesions and enhancing reflecting the essential features of metastatic behavior, we the prognosis. employed cDNA microarrays to compare the gene expression In this study we performed a statistical analysis of multiple profiles of L/B/K ALP- and CD99-transfected osteosarcoma gene expression datasets concerning the common biological clones showing low metastatic ability with those of osteo- problem of metastasis. Our comprehensive analysis includes sarcoma cell lines showing contrasting behavior. Changes the gene expression datasets derived from two experimental in gene expression were validated by real-time PCR and models previously obtained from the U-2 OS osteosarcoma immunohistochemistry in independent samples. In our study we cell lines characterized by a common reversal of malignancy identified several differentially expressed genes (GADD45α, and lack of metastatic phenotype. In particular, we have VCP, DHX9, survivin, α-catulin, ARPC1B) related to growth demonstrated in independent experiments that the forced arrest and apoptosis. Most of these genes are functionally expression of L/B/K ALP and likewise CD99 in U-2 OS related with the nuclear factor (NF)-κB cell survival pathway osteosarcoma cell lines markedly reduces the invasiveness that appeared to be inhibited in the less malignant osteosarcoma and metastatic ability of these cancer cells (4,5). Alkaline cells. Hence, we propose the inhibition of the NF-κB pathway phosphatases (ALPs) are a family of cell surface glycoproteins as a rational strategy for effective management of human that catalyze the hydrolysis of phospho-monoesters and release osteosarcoma. inorganic phosphate. Liver-bone-kidney (L/B/K) ALP, one of the four major isoenzymes belonging to this family, participates Introduction in bone mineralization. The precise biochemical function of bone ALP activity is still unknown, but in U-2 OS osteo- Metastasis is the dissemination of cancer cells from the primary sarcoma cell lines transfected with the full-length L/B/K ALP tumor to a distant organ and represents the most frequent cause gene a reduction in tumorigenic and metastatic ability has of death for patients with cancer (1). Osteosarcoma (OS) is a been observed associated with high levels of cellular activity of L/B/K ALP (4). CD99 is a transmembrane glycoprotein (6) _________________________________________ encoded by the MIC2 gene (7), which has been implicated in several cellular processes, such as cell adhesion, apoptosis and differentiation (8-12). Manara et al (5) have demonstrated Correspondence to: Dr Katia Scotlandi, Laboratorio di Ricerca that the forced expression of CD99 in two osteosarcoma cell Oncologica, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 lines significantly inhibits growth in anchorage independence Bologna, Italy E-mail: [email protected] as well as cell migration and leads to abrogation of tumorigenic and metastatic ability. Key words: gene expression profile, microarray, U2-OS, metastases, The fact that U-2 OS osteosarcoma cell lines transfected nuclear factor-κB with both L/B/K ALP and CD99 show identical behavior (that is, marked reduction of metastatic ability), makes these 17-31 10/12/07 14:53 Page 18 18 ZUCCHINI et al: APOPTOTIC GENES AS PREDICTIVE MARKERS IN OSTEOSARCOMA Table I. In vitro and in vivo biological features associated with malignancy of U-2 OS transfected clones. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Cell line In vitro parametersa In vivo behaviourb ––––––––––––––––––––––––––––––– –––––––––––––––––––––––––––––––––––––––––––––––– Soft-agar growth Migration Mice with tumor/total Mice with metastases/total ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– U-2 OS 742±102 215±40 7/10 (70%) 16/19 (84%) U-2/Empty 574±68 218±40 3/5 (60%) 9/10 (90%) U-2/ALP23 723±16 183±20 3/5 (60%) 10/10 (100%) U-2/ALP28 109±7c 26±5c 0/5 (0%) 3/7 (43%) U-2/ALP40 118±17c 92±6c 0/5 (0%) 4/14 (29%) U-2/CD99wt57 67±17c 90±18c 0/5 (0%) 0/10 (0%) U-2/CD99wt 136 124±26c 64±17c 0/5 (0%) 7/15 (47%) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– aSoft agar growth was measured by counting cell colonies 14 days after seeding of 10,000 cells in 0.33% agarose. Migration was assessed by counting the number of cells that migrated towards the filter to reach the lower chamber base of a Trans-well chamber. Cells (105) in IMDM plus 1% FBS were seeded in the upper compartment, whereas IMDM plus 10% FBS was placed in the lower compartment of the chamber as a chemoattractant. bTumorigenicity was determined after subcutaneous injection of 30x106 cells in female 4-5 week old Cr1:nu/nu (CD-1)BR athymic mice (Charles River Italia, Como). Experimental metastatic ability was evaluated after injection of 2x106 cells into a tail lateral vein. For detailed methods see Manara et al (4,5). cP<0.05, Student's t-test. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– cell lines a useful model to assess the intersection of multiple quality of RNA samples was determined by electrophoresis and independent gene expression signatures concerning the through agarose gels and staining with ethidium bromide; the common biological problem of dissemination. In particular, 18S and 28S RNA bands were visualized under UV light. To in our study we employed cDNA microarray technology to perform microarray hybridization, two independent extractions compare the gene expression profiles of L/B/K ALP- and of RNAs were performed from each cell clone. CD99-transfected osteosarcoma clones showing low meta- static ability with those of osteosarcoma cell lines showing cDNA microarray hybridization. Hybridizations were contrasting behavior, the purpose being to characterize a performed on Human 1 cDNA Microarray slides (Agilent common transcriptional profile reflecting the essential features Technologies, Inc., Palo Alto, CA) containing 16,281 cDNA of metastatic behavior. sequences. Total RNA was used to obtain labeled cDNA, according to the manufacturer's instructions (Agilent Direct- Materials and methods Label cDNA synthesis kit protocol, Agilent Technologies). cDNA from osteosarcoma cell lines transfected with L/B/K Cell lines. The parental osteosarcoma cell line U-2 OS was ALP and with CD99 was labeled with Cyanine 5-dCTP obtained from the American Type Culture Collection (Cy5), while parental U-2/OS cDNA was labeled with Cyanine (Manassas, VA). Cells overexpressing L/B/K ALP or CD99 3-dCTP (Cy3). In brief, 20 μg of total RNA was reverse- were obtained by using the calcium-phosphate transfection transcribed with a specific primer mixture using Moloney method previously characterized (4,5). Transfectants were murine leukemia virus reverse transcriptase (Agilent Direct- selected in 500 μg/ml neomycin (Sigma-Aldrich, St. Louis, Label cDNA synthesis kit) in the presence of Cy5 or Cy3 MO) and maintained in a selective medium for a maximum of (Perkin-Elmer Life Sciences Inc., Boston, MA) at 42˚C for 1 h. eight in vitro passages before in vitro and in vivo characteriz- RNaseI A (Agilent Technologies) was then added to degrade ation. In particular, U-2/ALP28, and U-2/ALP40, showing high the RNA. Labeled cDNAs were purified with the QIAquick levels of L/B/K ALP expression and activity, and U-2/CD99 PCR purification kit (Qiagen, GmbH, Germany). Additional clones (U2-CD99wt57 and U2-CD99wt136), overexpressing washes with 35% (w/v) guanidine hydrocloride were performed CD99 at the cell surface, were chosen for their low level of to ensure efficient removal of the unincorporated dye labeled malignancy. Table I summarizes the previously published nucleotides. Equivalent amounts of Cy5- and Cy3-cDNA
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